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C. R. Biologies 334 (2011) 737–741

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Comptes Rendus Biologies

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Animal biology and pathology/Biologie et pathologie animales

Blochmannia endosymbionts and their host, the ant Camponotus fellah:

Cuticular hydrocarbons and melanization

Relations entre endosymbiotes Blochmannia et leur hoˆte, la fourmi Camponotus

fellah avec hydrocarbures cuticulaires et me´lanisation

a,b, a a,

Danival Jose´ de Souza *, Se´verine Devers , Alain Lenoir *

IRBI, institut de recherche sur la biologie de l’insecte, UMR CNRS 6035, universite´ Franc¸ois-Rabelais, parc de Grandmont, 37200 Tours, France

Fundac¸a˜o Universidade Federal do Tocantins, Campus Universita´rio de Gurupi, Rua Badejo´s, 77402-970, Gurupi, TO, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: Carpenter ants (genus Camponotus) have mutualistic, endosymbiotic bacteria of the genus

Received 13 January 2011

Blochmannia whose main contribution to their hosts is alimentary. It was also recently

Accepted after revision 23 June 2011

demonstrated that they play a role in improving immune function as well. In this study, we

Available online 24 August 2011

show that treatment with an antibiotic produces a physiological response inducing an

increase in both the quantity of cuticular hydrocarbons and in the melanization of the

Keywords: cuticle probably due to a nutritive and immunological deﬁcit. We suggest that this is

Blochmannia

because it enhances the protection the cuticle provides from desiccation and also from

Cuticular hydrocarbons

invasions by pathogens and parasites. Nevertheless, the cuticular hydrocarbon proﬁle is

Antibiotic

not modiﬁed by the antibiotic treatment, which indicates that nestmate recognition is not Melanization modiﬁed.

Endosymbiotic bacteria

Carpenter ants

R E´ S U M E´

Mots cle´s : Les fourmis charpentie`res (du genre Camponotus) posse`dent des bacte´ries endosymbio-

Blochmannia

tiques mutualistes du genre Blochmannia qui contribuent a` l’alimentation de leurs hoˆtes. Il

Hydrocarbures cuticulaires

a e´te´ de´montre´ re´cemment qu’elles peuvent aussi favoriser les re´ponses immunitaires.

Antibiotiques

Dans cette e´tude, nous montrons qu’a` la suite d’un traitement antibiotique, les fourmis ont

Me´lanisation

une re´ponse physiologique induisant la production de plus d’hydrocarbures cuticulaires et

Bacte´ries endosymbiotiques

une cuticule plus me´lanise´e. Cela doit permettre une protection contre la perte d’eau et

Fourmis charpentie`res

pre´venir mieux contre l’intrusion de pathoge`nes et parasites. Ne´anmoins, le proﬁl

d’hydrocarbures cuticulaires n’est pas modiﬁe´ par le traitement antibiotique, ce qui

permet le maintien de la reconnaissance des conge´ne`res du nid.

ß 2011 Acade´mie des sciences. Publie´ par Elsevier Masson SAS. Tous droits re´serve´s.

1. Introduction studied thus far [1,2]. The function of endosymbionts has

not totally been elucidated, but their role in supplying

Carpenter ants (genus Camponotus) have established an complementary dietary nitrogen was shown through the

association with the intracellular endosymbiont Bloch- analysis of the genome sequence of two species of

mannia (g-Proteobacteria) found in all of the species Blochmannia [1,3–5] and in experiments eliminating the

bacteria through the administration of antibiotics and the

use of chemically–based diets [6,7]. However, the apparent

ability of the ants to survive without Blochmannia and the

* Corresponding author.

E-mail address: [email protected] (A. Lenoir). omnivorous behaviour of several Camponotus species

Author's personal copy

738 D. Jose´ de Souza et al. / C. R. Biologies 334 (2011) 737–741

suggests that the bacteria perform other functions for the 2.3. Chemical analyses

ants. We recently demonstrated that the presence of

Blochmannia may be important during the colony-found- Parts (i.e., the head and thorax, including the legs) taken

ing phase, and that these bacteria play a role in improving from 27 treated workers and 31 control ants were

the immune response of the ants [8]. immersed in 1 mL of pentane for 5 min and then removed,

Here, we tested other functions the bacterial endosym- and 5 ml of pentane containing 50 ng of eicosane (C20) was

biont carries out for the host ants. It is well known that added as an internal standard. For the analyses, the solvent

cuticular surface chemicals, mainly hydrocarbons, protect was evaporated until 5 ml remained which were then

insects from desiccation [9,10]. These hydrocarbons also injected into an FID gas chromatograph (VGM250Q

play an underestimated role in providing protection from system, Perkin-Elmer) using a DB-5 fused silica capillary

pathogens [11]. We tested the effects of an antibiotic column. The temperature was kept at 150 8C during the

treatment in eliminating or greatly reducing the quantity splitless initial 2 min, raised from 150 8C to 310 8C at 5 8C/

of endosymbiotic bacteria on the cuticles of the ants and on min and held at 310 8C for the last 10 min. The cuticular

the melanization processes. hydrocarbons had been previously identiﬁed [13,14]. To

also identify the smaller peaks, we analyzed the cuticular

proﬁle in greater detail with the same gas chromatograph

2. Material and methods coupled to a Perkin-Elmer mass spectrometer operating at

70 eV. We used a high temperature column to identify the

2.1. The ants peaks with more than 30 carbons (DB-5HT, 30 m,

0.251 mm Â 0.10 mm). The temperature program was

Incipient colonies of Camponotus fellah were collected 2 min at 100 8C; 6 8C/min until 350 8C and maintained

by Pr Abraham Hefetz in Tel-Aviv in March 2007. The for 5 min. The areas of the peaks were estimated through

colonies were placed into plastic containers peak integration using a TurboChrome Workstation. We

(20 Â 20 Â 10 cm) furnished with plaster nests and kept then calculated the quantities of substances using the

in a climate chamber (constant temperature of 28 8C, 12 h internal standard area (ng per thorax) and their relative

DL), and were fed twice a week with Tenebrio molitor larvae proportions. Hydrocarbon classes by percentage and total

and honey. Each colony contained one queen, and at least quantities were compared with the Mann-Whitney U test.

one hundred workers and brood. We used 10 control Data expressed as a percentage are used with the Arcsine

colonies (also fed with Tenebrio larvae and honey) and 10 (square root) transformation [15]. The proﬁles between

treatment colonies (fed with Tenebrio larvae and honey for the two groups were compared with a dendrogram using

the ﬁrst week, and then Tenebrio larvae and honey solution the single-link Ward method and Euclidian distance.

containing 1% of the antibiotic rifampicin for the second

week and thereafter). The treatment was applied during 3

3. Results

months. It has previously been shown that the antibiotic

treatment has no side effects on medium-sized ants (see

3.1. Degree of melanization

Discussion in [6,8]). For the experiments, we used

medium-sized foraging workers selected randomly from

Antibiotic-treated ants showed a signiﬁcant increase in

all of the colonies. We veriﬁed that there was no detectable

the degree of melanization when compared to the control

colony effect. In previous studies, we conﬁrmed the

group (Student’s t-test, t26 = 12.9, P < 0.00001; Fig. 1).

efﬁcacy of the antibiotic treatment in eliminating bacteria

Treated ants exhibited a degree of melanization approxi-

using real-time quantitative polymerase chain reaction

mately two times superior to that of control ants.

(PCR) and ﬂuorescence in situ hybridization (FISH) tests. It

was demonstrated that the antibiotic treatment reduced

3.2. Chemical analysis

the amount of bacteria by at least 75% [8].

We recorded 62 peaks, of which 58 were identiﬁed. Our

2.2. Degree of melanization of the cuticle

analysis was very detailed as 43 out of the 58 had not

We dissected the third tergite from 27 naive workers

and 29 workers treated with antibiotics. They were placed

in Clarion medium, and then mounted on glass slides.

Each piece was examined under a light microscope and

photographed using a digital camera (Olympus DP50). The

mean grey value of the cuticle fragment was measured

using ImageJ 1.37v software. The background grey value

was subtracted to correct the values of the fragments. The

mean grey value was obtained by measuring three square

plots in each ﬁgure. We assumed that the darkest grey

corresponded to the cuticle with the highest degree (i.e.,

totally black) of melanization. Melanization is normally

achieved a few days after imaginal eclosion, but changes Fig. 1. Means (Æ SE) of the degree of melanisation of the cuticle on control

occur in adults, for example, as they age [12]. (n = 27) and antibiotic-treated ants (n = 29). Student’s t-test, P < 0.0001.

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D. Jose´ de Souza et al. / C. R. Biologies 334 (2011) 737–741 739

previously been identiﬁed using earlier data [13], includ- process of parasites [17], and, therefore, that it increases

ing all of the components with more than 32 carbons not the ant’s immunological defense. We suggest that the ants

previously described with a total of 9% (Table S1 and Fig. S1 compensate for a deﬁciency in Blochmannia through

in the supplementary material). All were hydrocarbons physiological responses that increase their cuticular

except for a trace of one ester. There were three major melanization so as to be better protected from potential

peaks in the C27 series: C27 (peak n 11, mean 14.6 Æ SD invasions by pathogens and parasites.

o o

4.3%), 3-MeC27 (n 16, 25.3 Æ 2.9%) and 7-MeC27 (n 13, Our results also show that the ants’ cuticular hydrocar-

7.7 Æ 1.2%), and 16 peaks were comprised between 1 and 5%. bon proﬁle did not change after the antibiotic treatment.

All of the other peaks were less than 1%, and must be Although it was not quantiﬁed, observations of the ants

considered as traces and were detectable only in some suggest that nestmate recognition was not modiﬁed. As we

individual extracts due to the sensitivity of our gas previously observed [13], non-nestmates are normally

chromatograph (GC). rejected while nestmates are easily reintroduced into their

When comparing the proﬁles of treated and control ants, colony. Only one research study (on the termite Reticuli-

we did not ﬁnd qualitative differences; all of the substances termes speratus [18]) has indicated that there is a link

were present in both treated and control groups (all P between bacteria and nestmate recognition, but this is a

values > 0.25). The main classes for control ants are n- unusual case as nestmate recognition in these insects is

alkanes (25.30 Æ 6.22%), methyl-alkanes (49.56 Æ 3.41%), directly dependant on nutrition and bacteria are intestinal

dimethyl-alkanes (18.29 Æ 3.29%), trimethyl-alkanes guests. In C. fellah, the cuticular proﬁle is very stable; the

(3.88 Æ 1.12%) and a few alkenes (0.27 Æ 0.15%) (Fig. S2 and proﬁle remains similar for colonies collected during

Table S1 in the supplementary material). A dendrogram different years from the same site in Israel [13]. The

(Cluster analysis, Ward method, Euclidian distance) did not antibiotic treatment suggests that nestmate recognition in

separate the two groups; the ants were placed indifferently in these ants is not related to the bacterial endosymbionts.

the control or the treatment group according to their colony Since it has been shown that the quantity of the bacteria

(data not shown). Moreover, when we compared the decreases with the age of the ant [19], it seems logical that

percentages of the different substances, no signiﬁcant recognition occurs regardless of the presence of symbionts.

differences were detected, indicating that the chemical profiles If the cuticular hydrocarbon profile was not modified by

of control and treated ants were similar. These data indicate the antibiotic treatment, we did observe an increase in the

that the cuticular proﬁle of the ants did not change because of quantities of hydrocarbons. This is the ﬁrst time that a

the antibiotic treatment. Nevertheless, the antibiotic-treated change in the quantity of the cuticular hydrocarbons has

ants had a total quantity of hydrocarbons signiﬁcantly higher been correlated to an antibiotic treatment. Data on the

than the control ants (459.6 Æ 260 vs 300.5 Æ 159 ng/ant, quantities of hydrocarbons in ants are scarce. It is known,

respectively; P = 0.007, Mann-Whitney U test; Fig. 2). however, that the tropical ant Ectatomma ruidum has very

few (125–200 ng/worker) [20] and that Aphaenogaster

4. Discussion senilis has 2 to 4 mg per ant [21]; whereas Cataglyphis niger,

an ant that is well adapted to hot and dry climates, has 15

The treatment of the ants with antibiotics was to 50 mg per ant (i.e., 1/1000 of its body weight) [22]. The

correlated to an increase in the degree of cuticular composition and quantity of epicuticular lipids act to

melanization. This response can be linked to other waterproof the cuticle, and, therefore, play a role in

responses observed in a previous study conducted on preventing desiccation [23,24]. Long chain compounds are

C. fellah: the increase in the encapsulation response after generally thought to enhance desiccation resistance,

antibiotic treatment [8]. It is well known that a highly- although this is not always the case (reviewed by [9]

melanized cuticle can block microbial invasion [16]. It is and see [25]). Arthropods like desert Drosophila that dwell

also known that melanization is a part of the encapsulation in warm, dry environments tend to have hydrocarbons

with a longer chain length than their counterparts in more

mesic environments [26]. The environmental conditions

experienced by ants in different task groups may induce

changes in the cuticle. Workers that perform outside tasks

are more exposed to higher temperatures, lower humidity

and ultraviolet light. In the ant Pogonomyrmex barbatus, the

relative abundance of n-alkanes was 20% higher for

foragers and patrollers than for nest maintenance workers.

This may enhance the desiccation resistance of workers

exposed to a desert environment. However, in the study on

Pogonomyrmex, the foragers did not have longer chains

alkanes [27]. In the same species, the composition (i.e.,

percentage) for founding queens changed at the founding

stage, but the total quantity of hydrocarbons did not [28].

Changes in the hydrocarbon quantities need to be more

explained. It is not known, for example, if an increase in the

Fig. 2. Mean quantities of cuticular hydrocarbons on the thorax (ng/ thickness of the layer of hydrocarbons changes the

ant Æ SD), Mann-Whitney U test P = 0.008. protective properties of the cuticle.

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